AFM Imaging Suggests Receptor-Free Penetration of Lipid Bilayers by Toxins

Langmuir. 2019 Jan 15;35(2):365-371. doi: 10.1021/acs.langmuir.8b03146. Epub 2019 Jan 2.

Abstract

A crucial step of exotoxin action is the attack on the membrane. Many exotoxins show an architecture following the AB model, where a binding subunit translocates an "action" subunit across a cell membrane. Atomic force microscopy is an ideal technique to study these systems because of its ability to provide structural as well as dynamic information at the same time. We report first images of toxins Photorhabdus luminescens TcdA1 and Clostridium difficile TcdB on a supported lipid bilayer. A significant amount of toxin binds to the bilayer at neutral pH in the absence of receptors. Lack of diffusion indicates that toxin particles penetrate the membrane. This observation is supported by fluorescence recovery after photobleaching measurements. We mimic endocytosis by acidification while imaging the particles over time; however, we see no large conformational change. We therefore conclude that the toxin particles we imaged in neutral conditions had already formed a pore and speculate that there is no "pre-pore" state in our imaging conditions (i.e., in the absence of receptor).

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Bacterial Toxins / chemistry
  • Bacterial Toxins / metabolism*
  • Clostridioides difficile / chemistry
  • Exotoxins / chemistry
  • Exotoxins / metabolism*
  • Hydrogen-Ion Concentration
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism*
  • Microscopy, Atomic Force
  • Phosphatidylcholines / chemistry
  • Phosphatidylethanolamines / chemistry
  • Photorhabdus / chemistry
  • Protein Binding
  • Protein Conformation
  • Rhodamines / chemistry

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • Exotoxins
  • Lipid Bilayers
  • N-(lissamine rhodamine B sulfonyl)-1,2-dioleoylphosphatidylethanolamine
  • Phosphatidylcholines
  • Phosphatidylethanolamines
  • Rhodamines
  • toxB protein, Clostridium difficile
  • 1-palmitoyl-2-oleoylphosphatidylcholine